Pathological and Phylogenetic Characterization of Amphibiothecum Sp. Infection in an Isolated Amphibian (Lissotriton Helveticus) Population on the Island of Rum

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Pathological and phylogenetic characterization of Amphibiothecum sp. infection in an isolated amphibian (Lissotriton helveticus) population on the island of Rum

(Scotland)

CATERINA FIEGNA1, CHARLOTTE L. CLARKE1,2*†, DARREN J. SHAW1, JOHANNA L. BAILY 1, FRANCES C. CLARE 2, ALEXANDRA GRAY 2, TRENTON W. J. GARNER 2 and ANNA L. MEREDITH 1

1 Royal (Dick) School of Veterinary Studies & The Roslin Institute, University of Edinburgh, Roslin EH25 9RG, UK

2 Institute of Zoology, Zoological Society of London, Regent’s Park, NW1 4RY London, UK

SUMMARY

Outbreaks of cutaneous infectious disease in amphibians are increasingly being attributed to an overlooked group of fungal-like pathogens, the Dermocystids. During the last 10 years on the Isle of Rum, Scotland, palmate newts (Lissotriton helveticus) have been reportedly afflicted by unusual skin lesions. Here we present pathological and molecular findings confirming that the pathogen associated with these lesions is a novel organism of the order Dermocystida, and represents the first formally reported, and potentially lethal, case of amphibian Dermocystid infection in the UK. Whilst the gross pathology and the parasite cyst morphology were synonymous to those described in a study from infected L. helveticus in France, we observed a more extreme clinical outcome on Rum involving severe subcutaneous oedema. Phylogenetic topologies supported synonymy between Dermocystid sequences from Rum and France and as well as their distinction from Amphibiocystidium spp. Phylogenetic analysis also suggested that the amphibian-infecting Dermocystids are not monophyletic. We conclude that the L. helveticus- infecting pathogen represents a single, novel species; Amphibiothecum meredithae.

Key words: Amphibiocystidium, Dermocystidium, Amphibiothecum, palmate newts, infection, pathology, phylogenetics.

* Corresponding author: Royal (Dick) School of Veterinary Studies & The Roslin Institute, University of Edinburgh, Roslin, EH25 9RG, UK and Institute of Zoology, Zoological Society of London, Regent’s Park, NW1 4RY London, UK. E-mail: [email protected]

† Co-first authors.

INTRODUCTION In amphibians, Dermocystid infections manifest as spore-filled cysts Amphibian populations have seen a dramatic (Pascolini et al. 2003; Pereira et al. 2005; global decline (Blaustein and Wake, 1990, Raffel et al. 2008; González-Hernández et al. 1995; Houlahan et al. 2000; Roelants et al. 2010) that macroscopically present as small 2007; Blaustein et al. 2012), and amphibian discrete cysts (∼1 mm) to larger multi-focal infectious diseases are key factors implicated nodules. The limited data available suggest in amphibian population declines (Berger et that Dermocystid infection rarely causes al. 1998, 1999; Lips, 1999; Daszak et al. mortality in amphibian hosts or population- 2000). Most studies of amphibian infections level responses (Densmore and Green, 2007; focus on two pathogen groups – González-Hernández et al. 2010). However, chytridiomycete fungi (Lips et al. 2006; an association between the presence of Densmore and Green, 2007; Smith et al. Amphibiocystidium spp. and declines in 2009) and ranaviruses (Daszak et al. 2000; populations of Notophthalmus viridescens Duffus and Cunningham, 2010). However, a (Raffel et al. 2008) and Pelophylax rising number of reports of amphibian esculentus (Rana esculenta; Pascolini et al. infectious skin diseases have been attributed 2003) has been suggested. to a relatively poorly studied group of In 2006, an unusual skin disease was organisms belonging to the order reported in an isolated population of palmate Dermocystidia (Class Mesomycetozoea: newts (Lissotriton helveticus) on the Isle of Pascolini, et al. 2003; Raffel et al. 2008; Rum, Scotland (Gray, 2008), although González- Hernández et al. 2010; Rowley et anecdotal reports precede this (Isle of Rum al. 2013). The order Dermocystidia consists Rangers, personal communication, 2006). of pathogens known to infect mammals and There are two described Amphibiocystidium birds (Rhinosporidum sp.) (Herr et al. 1999; sp. in Europe; A. ranae known to infect green Vilela and Mendoza, 2012), fish frogs (Pelophylax lessonae and P. (Dermocystidium spp. and Rosette agent) esculentus) and a Dermocystid infection (Raganet al. 1996; Mendoza et al. 2002) and affecting L. helveticus in France (González- amphibians (Amphibiocystidium and Hernández et al. 2010). Skin lesions Amphibiothecum spp.) (Pascolini et al. 2003; affecting newts on Rum bear strong Feldman et al. 2005). Due to their similar resemblance to the latter. In both cases, morphology and explicit affinity to newts of the same species exhibited raised amphibian hosts, the amphibian-infecting cystic to nodular cutaneous lesions. Dermocystids were considered a single genus However, on Rum gross manifestations of (Pascolini et al. 2003). However, the use of disease can appear more severe than those more advanced molecular phylogenetics reported in France, and therefore, despite the recently saw the reclassification of these similarity, the pathogenesis and impact of pathogens into two distinct genera, this disease on the palmate newt population Amphibiocystidium and Amphibiothecum of Rum remains poorly understood. (Feldman et al. 2005), which include some of Anecdotal reports from Rum suggest that, the first pathogens described in amphibians whilst some newts succumb to severe (Perez, 1907, 1913), and are now known to infection, some populations are consistently be associated with both caudate and anuran free from infection (Anderson, 2010). Anti- species (Pascolini et al. 2003; Feldman et al. microbial peptides (AMPs) produced by 2005; Densmore and Green, 2007; Raffel et granular glands present in the skin of al. 2008). amphibians (Rollins-Smith et al. 2002; Zasloff, 2002) – part of the innate immune Mesomycetozoeans but distinct from other response – play an important role in the first Amphibiocystidium spp. However, he defence against microorganisms, and are emphasized the limitations of the small gene increasingly being linked to the resistance of region used for analysis and the small some species to skin infecting diseases, such number of known and sequenced as Chytridiomycosis (Woodhams et al. 2007; Mesomycetozoeans, stating that increased Rollins- Smith, 2009). It is conceivable that sequence data for the Dermocystids might gland function could extend to other fungal- improve the resolution and low bootstrap like organisms and gland structure should support (Feldman et al. 2005). reflect any such interaction (Zasloff, 2002). Here the combined results of gross, The classification of amphibian histological and molecular investigations are Dermocystids has largely been based on presented, characterizing a novel pathogen morphology (Perez, 1907; Granata, dermocystid-like infection causing cutaneous 1919; Poisson, 1937; Jay and Pohley, 1981, disease in palmate newts on the Isle of Rum, Pascolini et al. 2003). The unusual fungal- and providing phylogenetic support for the like nature of these organisms, and consideration of a new species within the differences in the terminology used in earlier genus Amphibiothecum; Amphibiothecum pathology descriptions, has led to uncertainty meredithae. We conclude that disease on in their taxonomic placement. For example, Rum represents a severe case of pathogens now considered to be from the Dermocystid-infection, and we examine the same genus have previously been classified preliminary investigation into the presence or as both protozoans (Dermosporidium spp.) absence of Amphibiothecum sp. and possible and fungi (Dermocycoides spp.) (Perez, morphological differences in granular glands 1907; Granata, 1919; Poisson, 1937; Jay and (linked to AMP production and host innate Pohley, 1981). DNA sequencing and immunity) that may play a role in the molecular phylogenetics have been important susceptibility of skin infection. in resolving the taxonomic relationships of these similar, Dermocystid-like pathogens MATERIALS AND METHODS described from amphibian hosts (Feldman et al. 2005; Pereira et al. 2005). Pereira et al. This study was carried out in May and June (2005) first used these techniques to analyse 2014 on the Isle of Rum (N57°00′55·1′′, a small region of 18SrRNA from infected P. W006°16′53·3′′) Scotland, the largest of the esculenta and P. lessonae. Small Isles in the Inner Hebrides. The island Recovered topologies confirmed the is of volcanic origin and contains numerous pathogen to be a member of the natural, dystrophic lakes and ponds that are Mesomycetozoeans, and further supported the natural habitat of the palmate newt (L. the creation of a single genus, helveticus), the sole amphibian species Amphibiocystidium (Pascolini et al. 2003), present on the island. to incorporate amphibian- infecting One hundred and sixteen live adult pathogens previously classified as newts were dip netted from three static water Dermocystidium, Dermocoides and bodies. The selection of sample sites was Dermosporidium (Pereira et al. 2005). based on a previous study (Anderson 2010) Feldman et al. (2005) later reclassified this and included one pond (control site) where group into two genera, Amphibiothecum and infection had not previously been recorded Amphibiocystidium, based on the placement and no macroscopic cutaneous lesions were of A. penneri as a member of the observed during our sampling (n = 12 newts). The remaining 104 newts were whole body were taken at predefined captured from the other two water bodies (n intervals; carcasses were sliced across the = 51 and n = 53). In addition, 23 dead newts head (rostral to the eyes and at the base of the were observed around the banks of one skull), and across the trunk (at the level of infected site. Forty newts (28 from infected the pectoral and pelvic girdles and with two ponds and 12 from a control site) were extra sections in between). The proximal retained and transported live to the field third section of the tail, including the cloaca, station where they were euthanized. To do and fore- and hind-limbs were longitudinally this, newts were fully immersed in an oriented. Tissues were sectioned at 5 μm aqueous solution of tricaine methane thick and stained with haematoxylin and sulphonate (MS-222, solution of 0·2% eosin (H&E) and examined by a light buffered with sodium bicarbonate) in microscope for the presence of parasitic accordance with the schedule 1 method under cysts. the Animals (Scientific Procedures) Act 1986 For transmission electron microscopy and the American Veterinary Medical (TEM) analysis, one formalin-fixed skin Association guidelines and recommendations tissue sample containing multiple for ethical euthanasia of amphibians subcutaneous cystic lesions was (AVMA, 2007). Dermocystid lesions have deparaffinized, post-fixed in 1% osmium previously been described on the skin of tetroxide and processed routinely by amphibian hosts, and on rare occasions on dehydration through graded acetones prior to the liver (Pascolini et al. 2003; Feldman et embedding in araldite resin. Ultrathin al. 2005; Raffel et al. 2008; González- sections (60 nm thick) were counter- stained Hernández et al. 2010). A detailed external with uranyl acetate and lead citrate and examination was performed where viewed with a Philips CM120 TEM. Images macroscopic dermal lesions were recorded, were taken on a GatanOrius CCD camera. detailing the size, colour and texture (Table To investigate the distribution and 1), as well as noting their abundance and condition of cutaneous granular glands, distribution across the newt body. In order to across diseased (n = 16) and control newts (n retain the anatomical location of visceral = 8), a standardized position was located in organs, a partial necropsy examination was the tail dorsum (a segment just caudal to the performed and the coelomic cavity was cloaca), that provided clear visibility of accessed via a ventral midline incision. The epidermal and dermal tissue in all newts and liver was fully exposed and, if present, cystic adequate coverage of granular glands. These lesions were recorded. Newts were then histological sections were photographed with individually fixed by full body immersion in a digital camera (Olympus DP72) at 4 × neutral buffered 10% formalin. A further six magnification (approximately 2 mm long newts with suspected Dermocystid-like segment). The number and diameter of all lesions were humanely euthanized by cutaneous granular glands in this section anaesthetic overdose as previously described were assessed using Cell^D software and were immediately stored in 100% (Olympus Soft Imaging Solutions). Granular ethanol for molecular analysis. glands were considered mature when more A subsample of 30 formalin-fixed than 3⁄4 of the gland alveolus was filled with newts, were processed for histological bright eosinophilic granular material. examination: 20 from infected ponds (18 with lesions and two without) and ten from the control site. Six axial sections of the Data analysis target amplicon to provide phylogenetic information. Both primers were compared A logistic regression analysis was performed with the online Basic Local Alignment in R version 3.2.1 (R Core Team, 2015) to Search Tool (BLAST) (NCBI, online), test the hypothesis that cutaneous granular confirming 100% homology with the glands of the dorsal tail were different in Mesomycetozoan species listed above and those animals with disease. The analysis was weaker homology with L. helveticus (84%, e- based on the infection status of the ponds value 0·026). Polymerase chain reactions (control, n = 8; infected, n = 16) using a (PCRs) were performed in 25 μL volume forward stepwise approach where the containing 1 μg DNA, 0·3 μM of each independent variables were: the number of forward (5′- GTAGTCATATGCTTGTCTC- cutaneous granular glands, their diameters 3′) and reverse (5′- and the relative percentages of full glands, TATTGCCTCAAACTTCCAT-3′) primer, were entered into the model with P ⩽ 0·1 and 200 μM dNTPs (Bioline, London, UK), and excluded with P⩾0·2 (Hosmer and 2·5 units of HotStarTaq Plus (Qiagen, Lemeshow, 2000). Crawley, UK). Two μL of each PCR product were electrophoresed on 0·6% agarose gel, Molecular phylogenetics stained with GelRedTM Nucleic Acid Gel A sample of oedematous tissue (1 mm2), a Stain (Biotium) alongside 2·5 μL of a 1 kb single dermal cyst or a single liver cyst was DNA HyperladderTM (BioLine) to assess excised from each of the six ethanol- amplicon size. Amplification products of the preserved newts, respectively. Excised tissue correct size were cleaned using polyethylene and cysts were washed in deionized water glycol precipitation, and commercially and dried. DNA was extracted using sequenced by GATC Biotech. Sequences DNeasy® Blood & Tissue Kit (Qiagen, were manually edited in BioEdit (Hall, 1999) Crawley, UK) according to the by trimming the outermost 5’ and 3′ ends manufacturer’s instructions. Primers specific near the sequencing primer site, where read to the mesomycetozoean clade were quality was poor or ambiguous. designed, targeting a 1400 bp region of 18s Edited sequences were aligned with rRNA, from an alignment of five all 26 18S rRNA mesomycetozoean Dermocystidia and Ichthyophonida 18s sequences submitted in GenBank. Non- rRNA sequences sourced from GenBank Mesomycetozoean outgroup sequences were (Dermocystidium sp. CM-2002, AF533950; chosen based on high query cover (98% Rhinosporidium seeberi, AF118851; ident.) but weaker similarity to the Rum Ichythophonus irregularis, AF232303; isolate than the mesomycetozoan sequences Ichthyophonus hoferi, U25637, (<93%); JN054684·1, DQ9958071·1, Pseudoperkinsus tapestis, AF192386). KC488361·1. Sequences were aligned using Sequences were aligned using ClustalW multiple sequence alignment in ClustalX2·1 (Larkin et al. 2007). Conserved regions were (Thompson, 1997). identified across the sequences to act as primers, ensuring enough variability in the

Maximum-likelihood (ML) analysis and MrModelTest (Posada and Crandall, 1998) Bayesian analysis have different strengths was performed in PAUP* 4·0 (Swofford, and may return different topologies due to 2002), testing 56 different models of DNA random errors in tree reconstruction evolution against a starting Neighbour- (Svennbland et al. 2006; Yang and Rannala, joining tree. The best-fit nucleotide model as 2012). For that reason, phylogenetic determined by the Bayesian information relationships were generated using both ML criterion (Schwarz, 1978; Ripplinger and and Bayesian analysis to compare the Sullivan, 2010) was Hasegawa–Kishino– topologies recovered and therefore offer Yano (Hasegawa et al. 1985) with gamma more support to the relationships and clades distributed rate variation and invariable sites recovered by both analyses. (HKY + G + I) ML analysis was performed in Paup*4·0 Ninety-three per cent (26/28) of (Swofford, 2002) setting parameter values as newts (15 males and 13 females) subject to detailed above, and specifying the outgroup. detailed external examination had Analysis was run with 1000 bootstrap macroscopic lesions of all types (e.g. A, B, C iterations of a heuristic search and tree- and D; Table 1). The shape of skin lesions varied depending on the number and size of bisection–reconnection branch swapping parasitic cysts and the associated (Posada and Crandall, 1998). So as not to subcutaneous oedema, whereas the shape of restrict model selection to named (i.e. Jukes parasitic cysts was consistently spherical and and Cantor) or pre- specified models, model pale grey/white. Additionally, subcutaneous averaging was implemented for Bayesian haemorrhage and circular skin ulcers (up to 6 analysis, sampling among the 203 general mm in diameter) were observed affecting 14 time reversible (GTR) models (Huelsenbeck (54%) newts; these were clustered over the et al. 2004). This was achieved by running tail, limb insertions and subgular regions. analysis using the reversible-jump Markov Parasite cysts and skin lesions were most chain Monte Carlo (rj-mcmc) in MrBayes frequently observed on the dorsal surface of 3·1·2 (Ronquist and Huelsenbeck, 2003), the body (n = 23). More specifically, cysts specifying gamma distributed rate variation, were located on the heads (n = 16), tails (n = 20), limbs (n = 20) and subgular regions (n = running two independent metropolis-coupled 18) along with solitary or multiple type A MCMC with four chains each, terminating lesions on the liver (n = 11). Four newts (two after 10 000 replications when the standard females and two males) (15%) had deviation between the split frequencies additional, severe and diffuse subcutaneous reached <0·01 (indicating that the trees being oedema associated with cutaneous sampled have onverged), sampling every depigmentation and presence of numerous 100. Final consensus trees were edited in type D lesions. These animals were moribund FigTree v1.4.2 (Rambaut, 2014) adding or showed limited body movements prior to bootstrap values or bipartition posterior euthanasia. probabilities. Histology and TEM RESULTS Histopathological examination confirmed the Gross pathology presence of parasite cysts, or related pathological changes in newts (n = 20) from A total of 66 newts with macroscopic infected ponds including the two individuals cutaneous lesions, suggestive of that had no macroscopic skin lesions. Dermocystid-like infection as described by Single or multiple, intact or ruptured González-Hernández et al. (2010), were spheroid parasite cysts were consistently recovered from two ponds; prevalence of present expanding the strata spongiosum and 47% [95%CI (34, 60)] and 76% [95%CI (64, compactum of the dermis (Fig. 1). Cysts 87)], respectively. Of the 23 dead newts were also present in the subjacent skeletal found, six were sufficiently well preserved to muscular layers (epi-and perimysium; n = 10 see that they had extensive dermal lesions, newts), oral mucosa (n = 5 newts), similar to those seen in live newts described gastrointestinal lumen (n = 1 newt), liver (n = hereafter. 7 newts) and cloaca stroma (n = 3 newts). See Supplementary Fig. 1A–D. Based on cyst morphology and the associated host’s inflammatory response (Fig. 2A–D), we could identify three different developmental stages: (1) developing (intact), (2) mature (intact and ruptured) and (3) degenerating and degenerated parasite cysts. All stages were observed concurrently affecting the same individual. Developing cysts were associated with no, or a mild, host cellular response (Fig. 2A), whereas ruptured and mature cysts were associated with tissue oedema and moderate to severe focal chronic-active inflammatory cell infiltrate. In addition, multinucleated Fig. 1. Intradermal Amphibiothecum sp. cysts in giant cells (foreign body-type cells) and focal palmate newt (L. helveticus). (A) Cross-section of the trunk. Microscopic appearance of multiple spheroid tissue necrosis (Fig. 2B) were occasionally cysts expanding and distorting the strata spongiosum present. In cases of severe subcutaneous and compactum of the dermis. Cysts are surrounded swelling, numerous and generally ruptured by diffuse and moderate subcutaneous oedema (*) parasitic cysts were present (up to 55), associated with mild mixed inflammatory infiltrates. surrounded by a moderate chronic-active Scale bar = 500 μm. Specific features: (C) cyst; (m) muscular fibres. inflammatory cell infiltrate and tissue oedema. Degenerating cysts (Fig. 2C) were comparatively smaller and were size from 250 μm to 1·7 mm in diameter characterized by a corrugated and convoluted separated from the host connective tissue by cyst wall, partially or fully detached from the 2–6μm thick eosinophilic cyst walls (Fig. host connective tissue. A focal chronic-active 3A). These contained a myriad of basophilic inflammatory host response surrounded developing immature endospores (IE). IE degenerating cysts and their lumen contained were polygonal to crescent shaped, measured an amphophilic to brightly eosinophilic approx. 6 μm in diameter and had granular matrix admixed with irregular amphophilic to basophilic, occasional islands of pale basophilic material vacuolated, protoplasm depending on the reminiscent of endospore formations. The stage of development. IE were packed within advanced stage of cyst degeneration round to oval and refractile (‘mucoid-like’) (degenerated cyst) (Fig. 2D) was chambers ranging in size from 10 to 40 μm in characterized by a focal granulomatous diameter (average approx. 24 μm), formed by lesion formed by packed mononuclear cells clusters of dividing and budding elements admixed with multinucleated giant cells, further separated by internal faintly centered on a remnant of collapsed cyst wall. distinguishable septa (Fig. 3B). Depending The majority of the parasitic cysts observed on the stage of cyst maturation, IE were in the liver (84%) were either ruptured or present along with a variable number of degenerated, and were surrounded by a dense mature endospores (ME); clusters of IE or chronic inflammatory cell infiltrate admixed ME were observed either at the center or at with several multinucleated giant cells the inner periphery of the cyst wall within the (foreign body-type cells) (Fig. 2E, F). same cyst (Fig. 3C). ME were located within Intradermal developing cysts less well-demarcated and comparatively appeared as spherical sporangia ranging in smaller internal chambers, were round to ovoid measuring on average approx. 15 μm the tail skin of infected animals were in diameter and were stained deeply classified as mature. Stepwise regression basophilic. In the later stage of cyst analysis indicated that infected newts had maturation, ME (hereafter granular mature marginally smaller glands than non-infected spore) were characterized by eosinophilic newts [(β = −0·025, 95% CI (−0·047, protoplasm swamped by numerous sub- −0·023), P < 0·031]. spherical (approx. 1 μm in diameter) basophilic granular bodies. Granular mature Molecular analysis spores were found within the lumen of mature cysts or were noted as free forms or Successful amplification of a 1400 bp DNA engulfed by macrophages in the contiguous fragment was achieved from all DNA host tissues (Fig. 3C and D). When observed extractions. Retrieved sequences from at higher magnification, granular mature samples representative of liver and dermal spore resembled clusters of merozoite cysts and subcutaneous oedema were elements budding from the surface of mature identical, confirming that each of these shizonts (Fig. 3D). No flagellae or other distinct pathologies is associated with the motility organelles were observed. presence of the same pathogen. These Ultrastructural (TEM) examination of an sequences shared high nucleotide similarity intradermal cyst revealed endospores as an (>94% similarity, >81% query cover) to individual unit enclosed in a thick electron members of the Mesomycteozoeans. Upon dense fibrous and granular matrix delimiting alignment with all Mesomycetozoean outer endospore capsule (Fig. 4). The 18srRNA sequences archived in GenBank, endospore protoplasm was further enclosed nearly complete consensus was observed (1 by an additional, non-uniformly distributed bp difference) between Rum and two granular coat formed by convoluted Dermocystid sequences isolated from membranes. The protoplasm of larger infected L. helveticus in France endospores was further subdivided through a (Dermocystid-Larzac; accession numbers process of invagination of these internal GU232542·1 and GU232543·1). delimiting membranes, where the outer Overall the topologies obtained by Bayesian matrix eventually enclosed them into distinct and ML techniques were congruent, although subunits. Dividing endospores contained some differences in the internal relationships between two to four daughter cells (Fig. 4A, between Dermocystidium sp. and insert). Amphibiocystidium sp. were recovered. Both Bayesian and ML analysis confirmed the Tail granular glands Rum parasite to be a member of the Dermocystids, forming a well-supported The number of cutaneous granular glands of clade with sequences from infected L. the dorsal tail skin in the control group (n = helveticus sampled in Larzac (Fig. 5: BS = 8) ranged from 7 to 14 (mean 10·4 ± 2·5). On 99·4%; PP = 0·7.) Whilst the Rhinosporidium average, 79% of tail glands were mature. sp. formed a monophyletic clade, the Gland diameters ranged from 53·3 to 401·0 amphibian-infecting Mesomycetozoeans μm (average 204 ± 80 μm). The number of were polyphyletic. glands in infected newts (n = 16) ranged from 8 to 28 (mean 13·4 ± 4·3) with their diameter ranging from 27·1 to 489·8μm (mean 147±90). On average 50% of glands in

Fig. 2. (A) Longitudinal section of the tail. A large single developing Amphibiothecum sp. cyst expands the stratum spongiosum of the dermis. The cyst lumen is filled with a myriad of basophilic IE. Note the absence of inflammatory cell infiltrate. (B) Cross-section of the trunk. The dermis and axial musculature are markedly expanded by the presence of multiple coalescing cysts. Focal necrosis and moderate chronic inflammatory cell infiltrate accumulates around (long arrow) or within (short arrow) the cysts. There is focal epidermal hyperplasia (arrowhead). (C) Degenerating intradermal cyst surrounded by chronic active inflammation (arrows). The cyst wall is distorted and partially detached from the host tissue by the presence of a clear space. Within the cyst lumen, embedded in amorphous eosinophilic matrix, there are isolated islands of polymorphic basophilic endospores and scattered endospores (arrowhead) which still retain the morphological features observed in developing and mature cysts. Scale bar = 200 μm. (D) Advanced stage of an intradermal degenerated cyst characterized by a granulomatous lesion. Multinucleated giant cells (arrow), plasma cells and reactive fibroblasts surround a collapsed, empty ellipsoid cyst. (E) Liver. Large hepatic granuloma consisting of a central empty cyst surrounded by concentric layers of proliferating fibroblasts forming a fibrous wall. Admixed there are macrophages, scattered lymphocytes and occasional multinucleated giant cells. There are subjectively increased numbers of melanomacrophages (arrows) within the surrounding hepatic parenchyma. (F) Liver. Hepatic granulomatous lesion. The cyst lumen is partially replaced by moderate number of foamy macrophages along with numerous multinucleated giant cells (arrows) and occasional granulocytes. Few granular mature spores are observed within the cyst lumen or within local macrophages. Inflammatory mixed cell infiltrate expands the surrounding oedematous hepatic parenchyma. Specific features: (C) Cyst; (ep) Epidermis; (g) Cutaneous granular gland; (ss) Stratum spongiosum of the dermis; (w) Cyst wall; (m) Skeletal muscular fibres. Scale bar A, B, E = 500 μm; C, D, F = 200 μm.

Fig. 3. (A) Cross-section of a developing intradermal Amphibiothecum sp. cyst containing myriad IE (IE). (B) High- power magnification of the inner lumen of a mature cyst. IE contained in septate chambers (arrows) clustering at the inner periphery of the cyst wall. (C) Cross-section of intradermal Amphibiothecum sp. cyst containing both IE and ME. Clusters of ME (long arrow) are opposite to IE (short arrow). Insert: ME and granular mature spores (circled). Note few macrophages containing intracytoplasmic granular mature spores surrounding the outer cyst wall (arrow). (D) Subcutaneous dilated lymphatic vessel adjacent to a ruptured cyst. Granular mature spores are free within the lumen (short arrows) or within macrophages (long arrows). Specific features are indicated with lower case letters: (c) Cyst; (ep) Epidermis; (g) Cutaneous granular gland; (ss) Stratum spongiosum of the dermis; (w) Cyst wall. Scale bar A, C = 200 μm; B = 20 μm; D = 50 μm.

Under both analyses a clade was recovered prevalence of infection across the island from suggesting a sister relationship between A. our sample of three water bodies, 63·5% of penneri and sequences from Rum and sampled live newts from infected ponds Larzac. Despite relatively weak confidence showed signs of disease, indicating that in this relationship (PP = 0·7; BS = 58·2%), infection is likely to be common in palmate support for a split between this clade and the newts on the island. rest of the Mesomycetozoeans was extremely As observed microscopically and high (BS = 100%; PP = 1·0.). ultrastructurally, intradermal parasitic cysts shared many common features with other DISCUSSION Dermocystic organisms (Broz and Privora, 1952; Jay and Pohley, 1981; González- This study reports combined gross, Hernández et al. 2010), in particular with that histopathological, ultrastructural (TEM) and described in palmate newts in France molecular findings that characterize (Gonzalez-Hernández et al. 2010). Similar to Amphibiothecum sp. infection in a their observation, we also noted the presence geographically isolated population of numerous endospores organized in of palmate newts on the Isle of Rum internal, septated chambers enclosed by a (Scotland). Whilst we can say little about the cyst wall.

Within the same cyst, clusters of endospores were observed at different developmental stages where the smallest compartmented chambers enclosed 2–4 single endospore elements. However, the arrangement of IE within mature cysts differed from that described by González- Hernández et al. (2010) where the authors propose a centrifugal fashion of endospores maturation. Instead, in this study, IE were present in clusters closer to the inner cyst wall of Fig.4. Transmission electron microscope microphotograph of Amphibiothecum sp. intradermal developed cysts and not exclusively at the cyst from an infected palmate newt. Multiple center of the cyst (Fig. 2). This finding endospores embedded in a thick electron dense fibrous therefore suggests a different pattern of and granular matrix (endospore capsule) (M). Variable endospore maturation and a potential sized food vesicles (long arrows) and multiple dense mechanism of endospores release. One round coarsely osmiophilic granular inclusion bodies (short arrows) are occasionally present in cells possibility is that fully matured endospores protoplasm. Membranous granular–fibrillar escape in a ‘programmed’ pattern as membranes (endospore membranes) forming described for R. seeberi (Mendoza et al. concentric rings around the protoplasm of each 1999), where endospores might develop individual endospore (m). The right lower insert toward the cyst’s pore from where they are shows one endospore dividing into four ‘daughter’ cells. Encircled one visible nucleus with prominent nucleolus.

Fig. 5. Consensus trees representing phylogenetic relationships of Mesomycetozoeans based on 18srRNA sequences. (A) Maximum likelihood analysis implementing HKY + G + I with node support shown using bootstrap support values; (B) Bayesian inference run using reversible-jump MCMC to average over the GTR models, with node support displayed as posterior probabilities; Amphibian-infecting species are highlighted in red, whilst sequences from Rum and Larzac are coloured blue. discharged. However, we were unable to restoration of surrounding tissues. Similarly, identify a cyst pore in any of the histological following the hepatic dissemination, sections. granulomatous lesions commonly occurred. In agreement with other amphibian- Although not the primary focus of this study, specific Dermocystid infections (Pascolini et we suggest that the cyst wall plays a crucial al. 2003; Pereira et al. 2005; Raffel et al. role in protecting the parasite from the host 2008; González- Hernández et al. 2010 and immune system during its development. In Courtois et al. 2013), diseased newts on fact, the virtual absence of an inflammatory Rum had macroscopic distinctive, raised, response surrounding intact developing cysts pale-white cystic skin lesions. In contrast to is noteworthy in comparison with the what was observed from Dermocystidim spp. inflammation surrounding ruptured and infection of Rana temporaria and P. degenerating cysts. Clinical manifestation of esculenta (Guyénot and Naville, 1922; Amphibiothecum spp. infection in palmate Pascolini et al. 2003) and Amphibiocystidium newts on Rum varied from subclinical (e.g. sp. infection in Eastern red- spotted newt apparently healthy individuals with only few (Raffel et al. 2008), here no ‘U’ or bent ‘C’ microscopic subcutaneous parasitic cysts), up shaped cysts were seen. In addition, we to severe generalized infection. While the frequently observed unusually large processes causing different clinical outcomes subcutaneous fluid filled vesicles/bulla (e.g. remain unclear, these observations suggest type D lesion) accompanied with full body that whilst some newts may recover from oedema as a result of Amphibiothecum sp. Dermocystid infection (e.g. presence of infection. microscopic dermal resolving lesions), other Similarly to Amphybiocystidium sp. individuals develop a generalized and infected newts in France (González- potentially fatal disease. Hernández et al. 2010), we observed skin Here we described animals with lesions primarily distributed over the newt microscopic parasite cysts, without the dorsum, limbs and tail, and only occasionally presence of gross lesions. González- over the ventral trunk. This is in contrast to Hernández et al. (2010) found no evidence of other Amphibiocystidium spp. infections asymptomatic or carrier-state individuals where cyst distribution was often from infected palmate newts in France; concentrated on the ventral surface (Broz and however, the presence of A. viridescens cysts Privora, 1952; Pascolini et al. 2003; Pereira was observed on the livers of apparently et al. 2005; Densmore and Green, 2007; uninfected Eastern red-spotted newts (Raffel Raffel et al. 2008). et al. 2008). This suggests that subclinical The presence of parasitic cysts at infection might be more common than different developmental stages and the expected and the observation of macroscopic related pathological changes (as observed skin lesions alone may not be a good proxy grossly and histologically) are together to determine infection prevalence. The suggestive of infection progression. While detection of pathogen genomic DNA from intact developing cysts were characterized by toe or tail clippings, and skin, oral or cloacal absent or a mild host inflammatory response, swabs, offer alternative detection methods fully mature and ruptured cysts were always extensively employed for Bd and Ranavirus associated with a discrete inflammatory surveillance (Annis et al. 2004; Hyatt et al. response. In the latter stages of cyst 2007; Skerratt et al. 2007; Goodman et al. degeneration, granuloma formation resulted 2013). However, the validity and accuracy of in reduced tissue inflammation and the these methods are being questioned. Whilst swabbing techniques have been found to fitness (e.g. reducing foraging and motility underestimate both infection prevalence and capabilities) or by rendering diseased newts parasite burden in Chytridiomycosis (Shin et more vulnerable to predation by al. 2014; Clare et al. 2016), they often miss compromising locomotion (Lindstrüm et al. subclinical Ranvirus infections (Greer and 2003). The presence of lesions in the oral Collins, 2007; Gray et al. 2012). Detailed mucosa could impact food intake, whereas histopathological examination therefore cysts and oedema on the cloaca could have represents an important diagnostic tool, an impact on breeding and courtship. If cysts particularly in cases of seemingly healthy and oedema on the cloaca are a common individuals with only subclinical disease. finding the ability of male to produce Severe full body oedema was spermatophores may be compromised. microscopically associated with a high Similarly breeding success may also be parasite burden along with a generalized reduced in other ways; male newts rely on form of infection evidenced by concur- rent tail fanning to entice females and lead them presence of cysts in the liver. To the authors’ over deposited spermatophors (Halliday, knowledge, this is the first published report 1990; Griffiths, 1996), behaviours that may of a generalized Dermocystid spp. infection, be hindered considerably by the presence of which results in severe subcutaneous oedema tail oedema or significant lesions. as confirmed by both histopathological and Since the majority of molecular analysis. Subcutaneous oedema Amphibiothecum spp. cysts were found would likely result from osmotic and within the dermis or adjacent tissues (e.g. electrolytic imbalances caused by extensive skeletal muscles bundles), parasite breaches in skin integrity due to numerous transmission is likely to occur by direct skin presence of parasite cysts. Parasite cysts in exposure to contaminated sediment/water or the liver might also result in hepatic infected newts. Infectious elements are likely insufficiency and systemic disease. to be released onto the skin surface and/or In addition to skin (mainly present surrounding environment after mature cysts within the dermis) and hepatic lesions, rupture as suggested by others (Perez, 1913; parasite cysts were also found in previously Broz and Privora, 1952; Jay and Pohley, unreported body sites such as oral mucosa, 1981). The transmission mechanisms of intestinal lumen, cloaca and infiltrating Dermocystidium spp. between their fish within the skeletal muscle bundles. hosts are well documented, where all species Secondary skin lesions were also produce zoospores to facilitate waterborne occasionally observed from infected newts transmission (Perkins, 1976; Olson et al. and consisted of skin ulcers and 1991). However, in this study, both haemorrhages as previously reported histological and TEM examinations (González-Hernández et al. 2010). We consistently showed no sign of parasite consider that secondary lesions resulted from spores developing flagellae. In addition to self-induced trauma, and from weakening or direct skin exposure, the possibility of oral breaches of the epidermis associated with transmission cannot be ruled out due to the parasite cysts. microscopic observation of parasite cysts Altogether these findings suggest that within the digestive system. This might also the intensity of infection may be critical in explain the presence of liver cysts, which determining the outcome of disease. Severe could pass through the bile duct following infection could increase mortality either ingestion as hypothesized previously (Raffel directly, or indirectly by compromising newt et al. 2008). However, due to the anatomical location of the liver, it is possible that sequences, and the short branch lengths parasite spores migrate through the sub- recovered under Bayesian analysis, are adjacent dermal-muscle layers and into the indicative of a single species. Whilst the liver. internal relationships between Further studies are necessary to Amphibiocystidium, Dermocystidium and confirm the mode of parasite transmission Rhinosporidium species were not consistent and also to investigate whether multiple across ML and Bayesian analysis, the infections, characterized by different amphibian and fish infecting pathogens are developmental cyst stages within the same not monophyletic. Rhinosporidium sp. individual, resulted from intra-tissue spread formed a discrete clade suggesting one of mature infectious elements (i.e. merozoite- evolutionary host-shift to mammalian hosts. type elements released from mature cysts), or However, the nested arrangement of repeated external exposure where each cyst Dermocystidium sp. and Amphibiocystidium represents a ‘discrete infection event’ as spp. suggests that pathogens can undergo proposed by Raffel et al. (2008). host-shifts, resulting in several, independent To explore the variation in disease amphibian- specific lineages. Whilst host susceptibility several studies have considered shifts between the lower invertebrates are not the role played by the innate immunity uncommon (Densmore and Green, 2007; provided by AMPs (Zasloff, 2002). Bandín and Dopazo, 2011, Price et al. 2014), Amphibian AMPs, released from granular the presence of multiple host-shifts from fish cutaneous glands are increasingly recognized to amphibians, but not the converse, is as a first-line of defence against pathogens atypical and appears to contradict previous that use the skin as their route of infection theories on host-shifts (Jancovich et al. (Rollins-Smith et al. 2002; Woodhams et al. 2010). In agreement with Feldman et al. 2006, 2007; Rollins-Smith, 2009). We (2005) our analysis distinguished A. penneri speculate that the reduced diameter of from other Mesomycetozoeans with high granular glands in infected newts, along with confidence, supporting its consideration as a a partial depletion of these glands, could separate genus. Sequences from Rum and have a nega- tive impact on the production of Larzac were also distinct from AMPs, resulting in a partially compromised Amphibiocystidium sp., instead forming a innate immune response and increased clade with A. penneri, a pathogen of B. susceptibility/severity to infection. Whilst we americanus in Northern America. The L. cannot allude to the mechanisms leading to helveticus infecting pathogens are therefore this difference, or the order of cause and not members of Amphibiocystidium, but effect, it is an interesting observation that instead should be consider a novel species may deserve more investigation. within the genus Amphibiothecum; Based on the high-sequence identity and Amphibiothecum meredithae. phenotypic similarities, the pathogen observed here is the same pathogen described ACKNOWLEDGEMENTS from L. helveticus in Southern France (González-Hernández et al. 2010). We would like to thank SNH for their support and guidance during field research. We gratefully Phylogenetic analysis not only emphasizes acknowledge all he staff of the Easter Bush their affiliation, but also highlights their Pathology unit at he Royal (Dick) School of distinctive- ness from other species of Veterinary Studies for their technical assistance; Dermocystid. The well- supported and in particular Ms Jennifer Harris for performing distinct clade containing the Rum and France microbiology tests and Dr. Sionagh Smith. We are also grateful to Stephen Mitchell (Kings Blaustein, A. R. and Wake, D. B. (1990). Declining Building) for assistance in TEM sample amphibian populations – a global phenomenon. preparation and for support with TEM. Trends in Ecology & Evolution 5, 203–204. Blaustein, A. R. and Wake, D. B. (1995). The puzzle of declining amphibian populations. Scientific American 272, 52– FINANCIAL SUPPORT 57.

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